We have been studying the spatiotemporal characteristics of the hemodynamic response function (HRF) to focal brain activity in marmosets (Callithrix jacchus), a small New World non-human primate that we believe is especially suitable to bridge the translational animal-to-human gap, and one in which transgenic techniques are becoming available to allow us to interrogate neurons and blood vessels in interesting and novel ways. We have trained marmosets to be imaged in the awake state and we are able to routinely obtain functional maps while the animal undergoes somatosensory, auditory, or visual stimulation. Complementary to fMRI, two-photon laser scanning microscopy (2PLSM) allows direct observation of neurons, astrocytes, microglia, and microvascular CBF. We developed 2PLSM techniques to allow visualization of neurons and blood vessels, and measured changes in neural activity and hemodynamics in the cortex of awake marmosets. We verified the optical imaging depth and clarity of the cranial window over 7 months, and optimized imaging parameters to achieve high spatial and temporal resolution. We developed algorithms to measure flow speed and diameter of individual blood vessels in real time, and the 3D topological connectivity of the vessels. We observed dense capillaries in the cortical tissues that connect from an arteriole to a venule in 15 branches. In order to label neurons underneath the cranial window, an intracortical injection of the genetically encoded calcium indicators GCaMP5G or GCaMP6s was given and functionality was verified in vivo by measuring the functional changes in fluorescence in response to somatosensory stimulation. The capillaries, along with the arterioles, dilate when an electrical stimulation was delivered to the wrist of the awake marmoset. Seven months after the viral delivery, 29% of GCaMP-expressing neurons are responsive to external stimulation of the somatosensory cortex. There are many advantages to using non-human primates to understand physiological and pathological processes in the highly evolved human brain and it is now possible to insert transgenes and to perform gene editing in primates, and we want to take advantage of these techniques to improve on our usage of marmosets to our specific goals. Our first priority was to generate transgenic marmosets expressing genetically encoded calcium indicators such as GCaMP5G/6s that will constitute a much-improved animal model for studying neurovascular coupling in relevant conditions. The rationale for first making a GCaMP-expressing marmoset comes from our desire to investigate neurovascular coupling at the spatial resolution of individual neurovascular units. To distinguish neurons, genetically encoded calcium indicators from the GCaMP family have been recently synthetized. When expressed in excitable cells of the brain, these molecules fluoresce upon calcium binding, becoming a visible marker of neural activity. We developed GCaMP5g and GCaMP6s expression constructs under the ubiquitous cytomegalovirus (CMV) and the human synapsin promoters. To generate GCaMP transgenic marmosets, we utilized high titer lentiviruses that expressed either GCaMP5g or GCaMP6s under the control of two different promoters: the human cytomegalovirus (CMV) enhancer/promoter for ubiquitous expression, or the human synapsin I (hSyn) promoter for neuron-specific expression. Marmoset embryos were collected from donor females either via nonsurgical uterine flushing of naturally fertilized (NAT) embryos, or alternatively via laparotomic follicular aspiration of unfertilized oocytes followed by in vitro maturation and fertilization (IVF). We performed 52 uterine flushing procedures and successfully collected 108 NAT embryos in 43 of them (82.7% successful flushing, 2.5 embryos/successful flushing). In addition, we collected 787 oocytes from 17 surgical laparotomies (average 46 oocytes/surgery). Of these, 625 (79.4%) successfully matured to the MII stage and, out of this cohort, 551 (88.2%) were successfully fertilized. Following embryo production and collection, 476 IVF and 100 NAT embryos were injected at their earliest possible embryonic stage with high titer lentiviral vectors. NAT embryos were transferred into recipient females on the same day, while IVF embryos were maintained in culture and examined for transgene expression 3-4 days after lentiviral infection. Among the injected IVF embryos, 385 (80.9%) developed successfully in vitro and, of those, 345 (89.6%) displayed fluorescence, indicating early expression of the transgene. To generate transgenic marmosets, 187 IVF and 88 NAT embryos were implanted into 51 and 31 recipient females, respectively. Twenty-two of the recipient females (26.8%, 22 out of 82; eight CMV-GCaMP5g, two CMV-mKO-GCaMP6s, six hSyn-mKO-GCaMP6s, two hSyn-GCaMP5g, three CMV-GCaMP6s, and one hSyn-GCaMP6s) became pregnant and 8 live newborns (one pair of twins, 6 singletons) were delivered naturally at full term. Among these 8 newborns, 3 died of unknown causes. One infant (TG-D2) survived for 15 days after birth, while the other two 113 (TG-D1 and D3) died within 24 hours after birth. The remaining 5 newborns, 3 males (TG-S, L and J) and 2 females (TG-Y and E) developed normally. At the present time, the two females reached sexual maturity, while the 3 males are still juveniles. From the 2 adult females we obtained oocytes and performed IVF, and were able to obtain 5 newborn infants of the F1 generation.